1. Field of the Invention
The present invention concerns an analytical method developed to accurately quantitate an analyte in the presence of an interfering substance.
2. Description of the Related Art
Antibodies have been widely used to detect and estimate specific antigens amidst the mixture of complex molecules in samples. The specificity of interaction with antigens has made them a valuable tool in such immunoassays. The assays enable the detection and quantitation of a wide variety of molecules such as hormones, tumor antigens, bacterial or viral antigens, etc. in biological samples. The range of such immunoassays has considerably broadened as a result of advances made in elicitation and characterization of antibodies against a variety of molecules such as proteins, peptides, carbohydrates, lipids, nucleic acids and small molecular weight compounds. The advent of monoclonal antibodies has added a new dimension by greatly enhancing the specificity of the immunoassays. The utility of the methods has further widened as a result of the flexible format (liquid vs. solid-phase) and various methods of enhancing the sensitivity of the detection. Amplification of the signal has been achieved using enzymes, radioisotopes and chemiluminiscence. Radioimmunoassay (RIA), Enzyme Linked Immunosorbent Assay (ELISA), immunohistochemistry and Western blot are just a few examples of the versatile nature and general applicability of immunoassays.
Immunoassays are used today not only in the diagnosis of infection by specific pathogens, but also for screening blood supply to ensure that they are free of blood-borne viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) etc. These assays are also widely used for the detection and/or pregnancy detection test, thyroid stimulating hormone (TSH), T3 and T4 for thyroid function test, human growth hormone (hGH) for various growth abnormalities, insulin for diabetes etc. Furthermore, immunoassays are also used to determine predisposition to certain conditions such as cancer. For example, serum level of prostate cancer antigen (PSA) is used to determine the onset of prostate cancer. Similarly, immunoassays also have great prognostic values. For instance, monitoring the level of HER2 expression in breast cancer patients is useful in predicting the success of a therapy based on the administration of HERCEPTIN® (Genentech, Inc., South San Francisco, Calif.), a recombinant humanized monoclonal antibody directed against p185HER2/neu, since this therapy primarily targets patients showing overexpression of this oncoprotein.
One of the problems frequently encountered in the use of immunoassay in estimating the amount of an analyte present in a sample is the presence of interfering substances that bind to the analyte, which makes a fraction of the analyte unavailable for reaction with antibodies against the analyte. The consequence of this interference is underestimation of the amount of analyte. There are several methods designed to address this problem. For example, one could separate the analyte and interfering substance present in the sample, for instance by chromatography, prior to subjecting the sample to immunoassay. For detection of microfilarial antigen in circulating immune complex from sera of filaria-infected individuals, Kobayashi et al. (Am. J. Trop. Med. Hyg. 57: 200-204 [1997]) used acidification of the test sera to pH 2 followed by restoring to pH 6 and then applying to an ELISA plate. The inhibiting moiety thus denatured by acid exposure may no longer be able to bind ligand, or may reform complexes slowly as compared to the capture of the ligand by the coated antibodies. Immune complex formation causes underdetection of p24 antigen in human immunodeficiency virus (HIV) infection. This problem was reportedly overcome by briefly boiling diluted plasma samples that released all complexed antigen, which was then measured by conventional method (Schüpbach et al., J. Infect. Dis. 170: 318-324 [1994]). Steindl et al. (J. Immunol. Methods 217: 143-151 [1998]) approached the same problem with slightly different solution; they used heat denaturation combined with exposure to sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) to successfully dissociate immune complexes. Similarly, exposure to a reducing agent (β-mercaptoethanol) and separation by free zone capillary electrophoresis was used to dissociate the aggregates of prion protein in order to estimate the level of free monomeric prions in bovine brain extract (Schmerr et al., J. Chromatogr. B. Biomed. Appl. 681: 29-35 [1996]). Another approach used to obviate this problem is described in a U.S. Pat. No. 5,501,983. In this instance, total prostate specific antigen (PSA) was estimated by separately measuring free PSA and PSA bound to proteinase inhibitor using two different assays. However, these approaches necessitate additional steps which are time consuming, cumbersome and often result in considerable loss of samples.
The present invention discloses a general strategy of accurately quantitating the amount of an analyte present in a fluid sample in the presence of an interfering substance. The strategy is exemplified by the application of this strategy for quantitative determination of serum HERCEPTIN® level in patients undergoing HERCEPTIN® therapy.
The ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4). The second member of the ErbB family, p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. Amplification of the human homolog of neu (i.e. HER2) is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No. 4,968,603). Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushigi et al., Mol Cell Biol., 6:955-958 (1986); Geurin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al, Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:354-357 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988); Williams et al. Pathiobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). ErbB2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31 (1993)).
Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of anti-ErbB2 antibodies one of which, called 4D5, inhibited cellular proliferation by 56%. A recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821,337) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al, J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the ErbB2/HER2 protein. Since HER2 is also overexpressed in other cancers, in addition to breast cancer, HERCEPTIN® holds a great potential in the treatment of such other cancers as well.
As described above, HERCEPTIN® has been successfully used in the treatment of breast cancer. However, the level of serum HERCEPTIN® should be maintained at 5-10 μg/ml during the therapy in order to effectively inhibit the growth of HER2 overexpressing breast cancer cells. Monitoring the level of serum HERCEPTIN® is thus crucial in the course of HERCEPTIN® therapy. A specific ELISA for the detection and quantitation of HERCEPTIN®, using HER2 extracellular domain (ECD) as a capture reagent, has been developed (Baselga et al., J. Clin. Oncol. 14:737-744 [1996]). However, the presence of circulating endogenous ECD in the serum of normal individuals, and often at high levels in breast cancer patients, interferes with the assay by competing with ECD coated on microtiter ELISA plates for binding to serum HERCEPTIN®. The net result is under-estimation of circulating HERCEPTIN® levels in serum. Therefore, there is a significant clinical need to improve the assay for accurately measuring serum level of anti-HER2 antibodies in the presence of circulating endogenous HER2 ECD.